Nucleic acid sequence analysis has become important in many research, medical, and industrial fields, e.g. Caskey, Science 236: 1223-1228 (1987); Landegren et al, Science, 242: 229-237 (1988); and Arnheim et al, Ann. Rev. Biochem., 61: 131-156 (1992). In large part, the strong interest in nucleic acid analysis has been driven by the development of several methods for amplifying target nucleic acids, e.g. polymerase chain reaction (PCR), ligation chain reaction (LCR), and the like, e.g. Kessler, editor, Nonradioactive Labeling and Detection of Biomolecules (Springer-Verlag, Berlin, 1992); Innis et al, editors, PCR Protocols (Academic Press, New York 1990); Barany, PCR Methods and Applications 1: 5-16 (1991). While such amplification techniques have the potential of providing highly sensitive and specific diagnostic assays, there is still a need to make assays utilizing such techniques convenient to perform in a clinical or field setting, especially when they involve the analysis of complex genetic systems, such as the extremely variable cystic fibrosis locus, or other highly polymorphic loci. In such systems, identifying the amplified product poses a special problem whose solution typically requires multiple post-amplification manipulations. A promising approach for identifying polynucleotides in such systems is the oligonucleotide ligation assay (OLA), Whiteley et al, U.S. Pat. No. 4,883,750. In this assay approach, oligonucleotides are prepared that are complementary to adjacent regions of a target sequence. The oligonucleotides are capable of hybridizing to the target so that they lie end-to-end and can be ligated when no mismatches occur at or near the contiguous ends. Whenever such mismatches do occur, then ligation is precluded. As a result, a set of oligonucleotide pairs may be provided which are perfect complements of all the allelic variants of interest at a given locus. By a judicious selection of labeling methodologies, a wide range of alleles, either from the same of different loci, can be specifically identified in a single assay.
Unfortunately, application of OLA to amplified target sequences complicates the assay, as exemplified by Nickerson et al., Proc. Natl. Acad. Sci. USA 87:8923-8927 (1990), which discloses the amplification of target DNAs by PCR and discrimination of variant DNA by OLA. After PCR amplification of the target DNA was performed in a first set of 96-well cluster plates, aliquots of the amplified samples were transferred to a second set of 96-well plates for OLA and the generation of ligation products. Aliquots of samples containing the ligation products were then transferred form the second set of plates to a third set of 96-well plates for detection of ligation products by an ELISA-based procedure.
The application of DNA-based assays employing amplification and OLA detection would be greatly facilitated if the number manipulations required to implement the assays could be reduced.